In this 5 year (R21/R33) project we will apply advanced proteomic and metabolomic nanoflow liquid chromatography-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry technologies in the study of both plasma and blood cells from individuals with normal glucose tolerance (NOT), impaired glucose tolerance (IGT), and recently diagnosed type 2 diabetes (T2DM). The overall approach endeavors to advance the study of Type 2 diabetes and pre-diabetes by identifying biomarkers at the level of the proteome and metabolome that are predictive of Type 2 diabetes and pre-diabetes in vivo. Our approach will utilize proteome-wide stable isotope labeling of peptides, as well as quantitative cysteine-peptide enrichment technology (QCET) and N-linked glycopeptide enrichment strategies to obtain broad proteome coverage and enhance quantitation. We will also utilize very low nanoflow LC separations to minimize ionization suppression and eliminate background ions originating from the solvent, thereby improving normalization of metabolite peak intensities and improve quantitation. This approach will be capable of rapidly identifying and measuring expression levels for thousands of peptides or concentrations of metabolites in a single analysis. Phase 1 of this project will (a) Define the sample processing and LC separation conditions necessary for broad proteome and metabolome coverage in human plasma and blood cell samples, (b) Perform a pilot study to define specific differences in the plasma proteome and metabolome of 10 individuals each with NGT, IGT, and T2DM, (c) Establish accurate mass and time tag databases for both peptides and metabolites detected in plasma and blood cells from individuals with NGT, IGT, and T2DM, and (d) Perform a pilot study to determine the feasibility of applying the nanoflow FTICR approach in the identification of non-enzymatically glycated plasma proteins. The refinement of this technological approach will provide the basis for high throughput studies of large numbers of samples. The application of this technology in Phase 2 of the project will involve (a) High throughput studies of large numbers of plasma and blood cell samples from individuals with NGT, IGT, and T2DM, (b) Validation of proteomic and metabolomic biomarkers of T2DM and pre-diabetes identified in Phase 1, and (c) Refinement of the sample processing and data analysis approach used in the identification of non-enzymatically glycated plasma proteins (Phase 1), in order to identify plasma proteins containing advanced glycation and/or advanced lipoxidation end products (AGEs/ALEs).